Coccidiosis is a disease of various animals in which the intestinal mucosa is invaded and damaged by a protozoa of the subclass Coccidia. The economic effects of coccidiosis can be especially severe in the poultry industry where intensive housing of birds favors the spread of the disease. Infection by coccidial protozoa is, for the most part, species specific. Numerous species, however, can infect a single host. For example, there are seven species of coccidial protozoa which infect chickens, six of which are considered to be moderately to severely pathogenic.
The life cycle of the coccidial parasite is complex. For example, protozoa of the genera Eimeria, Isospora, Cystoisospora, or Cryptosporidium typically only require a single host to complete their life cycle, although Cystoisospora may utilize an intermediate host. Under natural conditions, the life cycle begins with the ingestion of sporulated oocysts from the environment. When sporulated oocysts are ingested by a susceptible animal, the wall of the sporulated oocyst is broken in order to release the sporocysts inside. In poultry, the release of the sporocyst is the result of mechanical disruption of the sporulated oocyst in the gizzard. Within the sporocysts, are the sporozoites which are the infective stage of the organism. In poultry, the breakdown of the sporocyst coat and release of the sporozoites is accomplished biochemically through the action of chymotrypsin and bile salts in the small intestine. Once released, the sporozoites invade the intestinal mucosa or epithelial cells in other locations. The site of infection is characteristic of the species involved. For example, in the genus Eimeria, E. tenella is localized in the ceca; E. necatrix is found in the anterior and middle portions of the small intestine; E. acervulina and E. praecox occur in the upper half of the small intestine; E. brunetti occurs in the lower small intestine, rectum, ceca, and cloaca; E. mitis is found in the lower small intestine, while E. maxima can be found in any of these physiological locations.
Once inside the host animals' cells, sporozoites develop into multinucleate meronts, also called schizonts. Each nucleus of the meront develops into an infective body called a merozoite which enters new cells and repeats the process. After a variable number of asexual generations, merozoites develop into either microgametocytes or macrogametes. Microgametocytes develop into many microgametes which, in turn, fertilize the macrogametes. A resistant coat then forms around the resulting zygotes. The encysted zygotes are called oocysts and are shed unsporulated in the feces. Infected birds may shed oocysts in the feces for days or weeks. Under proper conditions of temperature and moisture, the oocysts become infective through the process of sporulation. Susceptible birds then ingest the sporulated oocysts through normal pecking activities or ground/litter foraging and the cycle repeats itself. Ingestion of viable, sporulated oocysts is the only natural means of infection.
Infection with coccidial protozoa results in immunity so that the incidence of the disease decreases over time as members of the flock become immune. This self-limiting nature of coccidial infections is widely known in chickens and other poultry. The immunity conferred, however, is species specific such that introduction of another species of coccidial protozoa will result in a new disease outbreak.
The oocyst wall of coccidial protozoa provides a highly effective barrier for oocyst survival. Oocysts may survive for many weeks outside the host. In the laboratory, intact oocysts are resistant to extremes in pH, detergents, proteolytic, glycolytic, and lipolytic enzymes, mechanical disruption, and chemicals such as sodium hypochlorite and dichromate.
Two methods are currently used to control coccidiosis in poultry. The first involves control by chemotherapy. Numerous drugs are available for the control of coccidiosis in poultry. Because of the number of species which cause the disease, very few drugs are efficacious against all species, although a single drug may be efficacious against several species. In modern broiler chicken production, for example, administration of drugs to control coccidiosis is routine. The expense for preventative medication against coccidiosis represents a significant cost of production.
Two programs of drug administration are commonly used in the domestic poultry industry. The simplest is the continuous use of a single drug from day one following hatching until slaughter. The second program is to use shuttle or dual drug program which involves the use of two different drugs, one administered in the “starter” ration and a second drug administered in the “grower” ration. This second method is often preferred as a method to minimize development of drug resistant strains of Coccidia. Using either method, drugs used are typically rotated two to three times per year in order to minimize the development of resistant strains.
The development of drug resistance by Coccidia is a serious limitation on the effectiveness of chemotherapy to control the disease. Surveys in the United States, South America and Europe have revealed widespread drug resistance in Coccidia. Since drug resistance is a genetic phenomenon, once established, drug resistance can remain in the population for many years until reduced by natural selection pressure and genetic drift.
The use of drugs in animals used for food production is also coming under increasing scrutiny by the public. Consumers are increasingly concerned with the possibility of drug residues in food. This creates pressure in the poultry industry to reduce the use of drugs to control coccidiosis.
Vaccination of birds against coccidiosis is an alternative to chemotherapy. An advantage of vaccination is that it can greatly reduce or eliminate the need to administer anti-coccidial drugs, thus reducing drug costs to poultry producers, preventing the development of drug-resistant strains, and lessening consumer concerns about drug residues.
Numerous methods have been developed to immunize poultry against coccidial protozoa. The successful methods have all been based on the administration of live protozoa, either fully virulent strains or attenuated strains. The most common route of administration is oral, although other routes have been used. Edgar, U.S. Pat. No. 3,147,186, teaches vaccination of chickens by oral administration either directly into the mouth or via the feed or water of viable E. tenella sporulated oocysts. Davis et al., U.S. Pat. No. 4,544,548, teaches a method of vaccination by continuous administration of low numbers of sporulated oocysts, with or without simultaneous administration of anti-coccidial drugs.
Oral administration of attenuated strains of sporocysts has also been utilized to confer immunity against coccidiosis. Shirley, U.S. Pat. No. 4,438,097; McDonald, U.S. Pat. No. 5,055,292; and Schmatz et al., PCT publication No. WO 94/16725. An alternative to attenuation is disclosed in Jenkins et al., Avian Dis., 37(1):74-82 (1993), which teaches the oral administration of sporozoites that have been treated with gamma radiation to prevent merogonic development.
Parenteral routes of vaccination have included subcutaneous or intraperitoneal injection of excysted sporozoites, Bhogal, U.S. Pat. No. 4,808,404; Bhogal et al., U.S. Pat. No. 5,068,104, and intra ovo injection of either oocysts or sporocysts, Evans et al., PCT publication No. WO 96/40233; Watkins et al., Poul. Sci., 74(10):1597-602 (1995). Sharma, J. Parasitol., 50(4):509-517 (1964), reported unsuccessful immunization trials involving intravenous, intraperitoneal, intramuscular, or subcutaneous injection of either viable oocysts or a mixture of oocysts, sporocysts and sporozoites. Thaxton, U.S. Pat. No. 5,311,841, teaches a method of vaccination against Coccidia by administration of oocysts or sporozoites to newly hatched chicks by yolk sac injection.
Regardless of the route of administration, procedures for the production of coccidiosis vaccines are quite similar. Briefly, coccidial protozoa are produced by infecting host animals with a single species of coccidial protozoa. These “seed stocks” are often clonal in nature, that is, derived from a single organism in order to insure the presence of only the species of interest. Seed stocks may be wild type, that is, isolated from the field, or they may be precocious or attenuated strains. The protozoa are then allowed to undergo replication in the host, after which, protozoa are collected from the animals, usually from the excreta. The use of attenuated strains typically results in fewer shed oocysts from the host animal. The protozoa are then separated from the excreta by well known techniques such as salt floatation and centrifugation. At the time of collection, the protozoa are at the non-infective oocyst stage of the life cycle. In order to become infective, and therefore useful for vaccines, the oocysts must be induced to undergo sporulation. In members of the genus Eimeria, sporulation typically involves the incubating the oocysts in a 1% to 4% aqueous solution of potassium dichromate at 19° C. to 37° C. with constant aeration. Data on oxygen consumption are conflicting, with Smith and Wilson (J. Parasitol. 30:295-302, 1944) reporting increased oxygen consumption for E. tenella and Wilson and Fairbairn (J. Protozool. 8:410-416, 1961) reporting no change in oxygen consumption for E. acervulina. Sporulation is usually complete within 12 to 24 hours depending on the temperature used. Monitoring of the sporulation process is accomplished by microscopic examination of the protozoa. Storage compositions found in the prior art typically include an aqueous solution of potassium dichromate. The sporulated oocysts are usually stored in 1 to 4% aqueous solution of potassium dichromate to prevent bacterial growth, however, other storage media have been used.
Current vaccines available for the prevention of coccidiosis typically contain a 2.5% weight to volume solution and contain approximately 1,600 oocyts per dose (400 sporulated oocysts representing four different species). The current commercially available vaccines contain from about 1.6×10−2 μg of potassium dichromate per oocyst to about 0.16 μg of potassium dichromate per oocyst.
Although widely used for sporulation and storage, potassium dichromate has several properties which make its elimination from biologicals highly desirable. Potassium dichromate is a strong oxidizer and has been reported to affect the respiratory system, liver, kidneys, eyes, skin and blood. It is a known carcinogen and upon disposal is regarded as a hazardous waste. Because of its high toxicity, compounds containing potassium dichromate are particularly unsuitable for parenteral administration. Thus, it would be highly advantageous to eliminate potassium dichromate from the production and storage of materials to be administered to animals, especially food animals.